1. Field of the Invention
The invention relates to contrast agents and methods of preparation thereof for use in various imaging modalities, and/or for use in therapy.
2. Description of Related Art
Introduction to Imaging Modalities
Various in vivo imaging processes, including ultrasound, magnetic resonance and computed tomography, are used as medical diagnostic tools. The underlying principle of each imaging modality is generally that the differences in a particular property or properties (e.g., acoustic properties, proton density, etc.) of the organs, tissue and other substances within the body at a location to be examined are detected and then translated into an image. The various modalities, however, rely on very different principles to generate images. The effectiveness of any of these imaging processes, and the resolution of the resulting images, in a great part depends on the degree of contrast between the body parts that the imaging equipment is able to detect so as to delineate the features of the region of interest within the subject body area. As a result, use of internally administered agents specifically designed to enhance the degree of contrast detected with a particular modality has become common. The differences in the imaging techniques involved with various modalities, however, have thus far generally restricted the use of any particular contrast agent to one imaging modality.
Ultrasound
Ultrasound (xe2x80x9cUSxe2x80x9d) is an imaging process that relies on the reflection of sound waves within the body to produce an image thereof. High frequency sound (ultrasonic) waves, which are above the range of sound audible to humans, are directed at the region of interest within the body. The waves are reflected back wherever there is a change in the physical parameters of the structures within the body, e.g., a change in density between two adjacent organs. The ultrasound equipment receives the reflected sound waves and transmits them into an image based on the differing levels of intensity of the reflected waves.
Use of a contrast agent enhances the differences in intensities of the reflected waves. For example, intravenous encapsulated microbubble contrast agents have become an established clinical tool for enhancing medical diagnostic ultrasound and Doppler sensitivity. Some current contrast agents function to enhance the appearance of the blood pool and to define its architecture and integrity. Other contrast agents provide passive, targeted, organ-specific imaging based upon the bio distribution and pharmacokinetics of the circulating contrast agent, localizing in, for example, the liver, spleen, kidney and lung.
The interaction of encapsulated microbubble contrast agents with ultrasound is complex. The microbubble response relative to a driving acoustic pressure can be divided into three categories: (1) linear scattering, (2) nonlinear scattering, and (3) cavitation/destruction. Microbubbles produce linear scattering with low acoustic driving pressures and produce non-linear scattering with moderate acoustic driving pressures. At moderate acoustic driving pressures, microbubbles exhibit pressure peaks at the compressional phases of the source thereby providing both harmonic and subharmonic energy greater than the surrounding medium. At very high acoustic driving pressures microbubbles cavitate or destruct as a result of fragmentation and deflation and thus create an associated acoustic emission signal. The absolute values for low, moderate and high acoustic driving pressures are not well defined and depend upon not only the acoustic parameters of the ultrasonic source but also the constituent physical properties of the microbubbles themselves, as well as the fluid surrounding them.
A significant problem with the use of microbubble contrast agents result from the machinery associated with the imaging process. Typical medical diagnostic ultrasound imaging machinery produces acoustic pressures that can range from 0.5 to 3 mega pascals (MPa). This acoustic pressure range can destroy some microbubble contrast agents during the imaging process, thus reducing the efficacy of the contrast agent and also reducing the effective imaging time (half-life) of the contrast agent.
Albunex(copyright) (from Molecular Biosystems, of San Diego, Calif.), the first commercially available ultrasound contrast agent, is a suspension of air-filled albumin microspheres produced by sonication of a heated solution of 5% human albumin. The major drawbacks associated with use of Albunex(copyright) as a contrast agent for ultrasound are its short plasma half-life and its acoustic instability relative to pressure changes. The plasma half-life of radiolabeled Albunex(copyright) microbubbles after intravenous injection is less than one minute. In addition, backscatter intensity falls as pressure rises, an effect that has been demonstrated in vivo as a systolic fall in videointensity following intravenous injection. Moreover, albumin microbubbles cannot by used with other modalities such as magnetic resonance imaging or computed tomography because the microbubbles do not have the functional characteristics required for such modalities.
With the development of medical ultrasonic contrast agents, the theoretical behavior of encapsulated microbubbles has generated substantial interest. Ye found that at frequencies below or slightly higher than the resonance, acoustic scattering by Albunex(copyright) bubbles is nearly omni-directional and bears similarities to that by usual air bubbles. (Ye, xe2x80x9cOn Sound Scattering and Attenuation of Albunex(copyright) Bubbles,xe2x80x9d J. Acoust. Soc. Am., 100(4) 2011-28, (1995)). The Ye reference also reveals that the scattering by Albunex(copyright) bubbles can be highly anisotropic when the frequency is above resonance. Work by de Jong showed large differences in non-linear behavior between ideal and Albunex(copyright) microspheres due to the additional restoring force and friction inside the shell that surrounds the Albunex(copyright) microsphere. (de Jong et al, xe2x80x9cHigher Harmonics of Vibrating Gas-Filled Microspheres, Part One: Simulations,xe2x80x9d Ultrasonics, 32(6) 447-453 (1994)).
Prior efforts to address the need for an increase in the plasma half-life of medical ultrasonic contrast agents have focused on: (1) strengthening the structure of the encapsulating shell, (2) employing different substances for the encapsulating shell, or (3) chemical modification of the microsphere surface, for example, by pegylation. For example, the use of galactose with human serum albumin microspheres appears to strengthen the shell, thereby increasing the half-life to 3 to 6 minutes. (Goldberg, xe2x80x9cUltrasound Contrast Agents,xe2x80x9d Clin. Diag. Ultrasound, 28:35-45 (1993)). Kimura et al. utilized small unilamellar vesicle (xe2x80x9cSUVxe2x80x9d), large unilamellar vesicle (xe2x80x9cLUVxe2x80x9d) and multilamellar vesicle (xe2x80x9cMLVxe2x80x9d) as echogenic liposomes. (Kimura et al., xe2x80x9cPreparation and Characterization of Echogenic Liposome as an Ultrasound Contrast Agent,xe2x80x9d Chem. Pharm. Bull., 46(10) 1493-96 (1998)). The acoustic reflectivity obtained with the echogenic MLV was larger than that of the gas bubbles enclosed within a surfactant mixture. A half-lifetime of 39 minutes was observed for the MLV prepared from egg-yolk phosphatidylcholine liposomes. The duration of reflectivity was prolonged drastically to a half-lifetime of 866 minutes by incorporating cholesterol into the MLV, although, significantly, the echogenicity was decreased by such incorporation. Although there have been a number of important steps at lengthening the effective imaging half-life of injectable ultrasonic contrast agents using liposomes, there has been an overall reduction in the echogenicity of these agents.
Thus, although there are a number of ultrasonic contrast agents now available commercially, and despite significant research directed to many of these agents, limitations still exist with these agents. Furthermore, few ultrasonic contrast agents can be used with other imaging modalities.
Magnetic Resonance Another imaging technique is magnetic resonance (xe2x80x9cMRxe2x80x9d) imaging. This modality relies on detecting the emission of electromagnetic radiation by certain atomic nuclei in the body upon application of pulsed radio frequency signals in the presence of a magnetic field. The resulting magnetic echoes produced when the signal is terminated ultimately are translated into an image.
Use of certain contrast agents with MR is known in the art. Contrast agents are commonly used intravenously to change the local magnetic field in tissue. Generally, abnormal tissue will respond differently in the presence of the contrast agent as compared to normal tissue and will give off a different magnetic echo. Thus, when the magnetic echoes are translated into an image, an image of the tissue abnormalities is provided.
The use of gadolinium oxide (Gd2O3) particles alone measuring less than 2 micrometers (xcexcm) in diameter as a prototype MR contrast agent has been examined for imaging the liver and spleen. (Burnett et al., xe2x80x9cGadolinium Oxide: A Prototype Agent for Contrast Enhanced Imaging of the Liver and Spleen with Magnetic Resonance,xe2x80x9d Magnetic Resonance Imaging, 3:65-71 (1985)).
Another study evaluated the effects of gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA), albumin Gd-DTPA, and Gd2O3 on imaging of the spleen and renal cortex. (Daly et al., xe2x80x9cMR Image Time-Intensity Relations in Spleen and Kidney: A Comparative Study Of GdDTPA, Albumin-(GdDTPA), And Gd2O3 Colloid,xe2x80x9d American Journal of Physiologic Imaging, 5:119-24 (1990)). The suspension of Gd2O3 used in the studies by Bumett and Daly was synthesized by titrating a GdCl3 solution with NaOH. With this method of preparation, residual GdCl3 is likely to remain in the Gd2O3 preparation, such that extreme toxicity from inadvertently incorporated free GdCl3 is possible. With most chelated gadolinium contrast agents, only one gadolinium atom per molecule is present in commercially-available contrast media manufactured for use in MR imaging, so that the enhancement capabilities of the contrast agent are limited. In addition, synthesis of albumin particles and also albumin microspheres tagged with gadolinium chelates on the surface would also be expected to have decreased MR sensitivity due to the limited number of sites for conjugation of the gadolinium chelate to the microsphere surface.
Magnetite (Fe3O4) albumin microspheres (xe2x80x9cMAMxe2x80x9d) have been used as a superparamagnetic contrast agent for reticuloendothelial MR imaging. (Widder et al., xe2x80x9cMagnetite Albumin Suspension: A Superparamagnetic Oral MR Contrast Agent,xe2x80x9d ARJ, 149: 839-43 (1987)). MAM was synthesized by combining 5% human serum albumin (xe2x80x9cHSAxe2x80x9d) and magnetite to create albumin microspheres using a modified water-in-oil emulsion polymerization technique. Nonlinear behavior of MAM with increased applied external magnetic field over 0.3-0.9T was observed. The influence of magnetite on T2 relaxation is believed to be due to local field inhomogeneities generated by the large magnetic moment of Fe3O4, which causes dephasing of proton spins and an acceleration of T2 relaxation with negligible T1effects. Because iron oxide is predominately a T2 relaxation agent, MAM has limited usefulness in conventional MR imaging. Additionally, based on the lower density of iron oxide relative to other heavy metals, iron oxide, and thus MAM, has a very limited utility for other imaging modalities, such as computed tomography.
As with contrast agents for US, contrast agents for MR also have limitations, both when used with MR and if used with other imaging modalities. Few MR contrast agents have even been evaluated for use with other imaging modalities.
Computed Tomography
Computed tomography (xe2x80x9cCTxe2x80x9d), also called computerized axial tomography, is an imaging modality that utilizes a toroidal, or donut-shaped x-ray camera to provide a cross-sectional image of the body area of interest. Use of certain contrast agents to improve CT images is known. Generally, the contrast agent localizes in a particular body compartment and differentially opacities normal or abnormal tissue. The contrast agent causes the tissue to inhibit passage of x-rays to produce a shadow of positive contrast in the resulting image. Iodine-based contrast agents are considered to be the industry standard with CT.
Gd-DTPA contrast agents have been used for certain limited applications in conventional angiography and CT imaging. (Bloem and Wondergem, xe2x80x9cGd-DTPA as a Contrast Agent in CT,xe2x80x9d Radiology, 171:578-79 (1989)). A major drawback associated with using Gd-DTPA contrast agents for CT imaging is the fact that only one electron dense (gadolinium) atom per molecule is present in commercially-available contrast media. In comparison, two widely used contrast agents, Optiray(copyright) (by Mallinckrodt, Inc., of St. Louis, Mo.) and Ultravist 300(copyright) (by Berlex Laboratories, Inc., of Wayne and Montville, N.J. and Richmond, Calif.), contain three electron dense (iodine) atoms per molecule. In addition, the molar concentration of gadolinium in commercially-available gadolinium-based contrast agents, such as Magnevist(copyright) (by Berlex Laboratories, Inc., of Wayne and Montville, N.J. and Richmond, Calif.), is 0.5 mol/L, which is one-fifth the molar concentration of iodine in Optiray(copyright) (320 mg of iodine per mL, or 2.5 mol of iodine per liter). Thus, presently available MR contrast agents provide sub-optimal CT enhancement and/or are not well-suited for use with other imaging modalities, such as CT and US.
Study Of Contrast Agents In Different Imaging Modalities
To date, few contrast agents have been used for imaging studies utilizing multiple imaging modalities. Correlative studies using combinations of imaging methods, most notably CT and MR imaging, are frequently performed in order to improve the accuracy of diagnosis or assess the efficacy of treatment routines. Magnevist(copyright) (Gd-DTPA) and a few other gadolinium-containing MR contrast agents have been used for this purpose, but limitations associated with the dosage and cost of commercially available MR contrast agents have prevented widespread use. Further, these agents would confer no obvious benefit to US imaging due to their low compressibility and the high concentrations required in order to provide effective US imaging.
Perfluorocarbon emulsions have been evaluated for contrast image enhancement. Perflubron (perfluorooctyl bromide, xe2x80x9cPFOBxe2x80x9d) emulsified with egg yolk lecithin has been tested for use in US (due to its high density), MR (fluorine nuclei imaging or as a signal void for hydrogen nuclei imaging) and CT imaging (due to its bromine atom). However, neither fluorine MR imaging nor signal void imaging have found widespread use in hospital or clinical practice, where T1, (and to a lesser extent, T2) imaging of protons is typical. Also, PFOB is less dense radiographically, i.e. less radio opaque than iodine-based CT contrast agents, making larger doses necessary in order to achieve adequate x-ray attenuation.
Despite the significance of contrast agents in medical diagnostics and the ever-present need for correlative studies, no single commercially-available contrast agent provides effective, cost-efficient image enhancement utilizing more than one imaging modality.
The invention relates to a new class of contrast agents, namely paramagnetic protein microspheres, for use with multiple imaging modalities. More particularly, this invention relates to gadolinium oxide albumin microspheres (xe2x80x9cGOAMxe2x80x9d), in both unmodified and surface-modified (including pegylation, antibody attachment, etc.) forms, that are used as contrast agents with the more widely used imaging modalities, including US, MR, and CT. In a preferred embodiment, Gd2O3 molecules are encapsulated in albumin microspheres. Unmodified and/or surface-modified GOAM of the present invention can function as contrast imaging agents for multiple imaging modalities, such as US, MR and CT.
With respect to US, these microspheres generally have the potential to withstand greater acoustic pressures than prior contrast agents due to the synthesis method used in the present invention. The presence of Gd2O3 sequestered within albumin microspheres significantly enhances echogenicity of the protein microspheres. The increased functionality of the GOAM of the present invention as a US contrast agent derives from increased echogenicity due to the effect of Gd2O3 on density, compressibility, absorption cross-section, scattering cross-section, and velocity of sound of the albumin microspheres. Additionally, toxicity may be decreased because the overall Gd2O3 concentration required for ultrasound image enhancement is reduced due to gadolinium oxide being sequestered within albumin microspheres.
The GOAM of the present invention also can provide enhanced CT imaging due to the high atomic weight and high k-edge of gadolinium. Additionally, GOAM contains multiple Gd2O3 particles, each of which are made up of several gadolinium atoms, improving the utility of GOAM as an x-ray attenuation agent for CT.
T1, and T2 relaxation enhancement in MR imaging is due to the paramagnetic properties of gadolinium, whose seven unpaired electrons account for its high relaxivity, and the super-paramagnetic and/or ferromagnetic properties of Gd2O3, which will be non-specifically sequestered in albumin microspheres, thereby allowing for increased interaction with mobile protons, the potential for relaxation via physical rotation of Gd2O3 and a decreased tumbling rate of Gd2O3 when associated with albumin microspheres. In addition, improved T1, and T2 relaxation at lower concentrations of Gd2O3 is anticipated due to the association of Gd2O3 with a macromolecule, i.e. an albumin microsphere.
GOAM also may be used in therapeutic applications, such as gadolinium neutron capture therapy, because of the high cross-sectional density and high neutron capture rate of gadolinium. Gadolinium has the highest thermal neutron capture cross-section of any known element. GOAM also may be used to encapsulate other therapeutic agents, such as antineoplastic drugs.